This work was supported by National Institutes of Health (NIH) grant R01 CA222560 and by Department of Defense Breakthrough Award W81XWH-18-1-0037.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Brigham and Women&#8217;s Hospital.
1. Generation and maintenance of floxed mice
<ol>
	<li>Obtain breast cancer-relevant floxed conditional knockout (e.g., Trp53tm1Brn [referred to as Trp53L/L], Brca1tm1Aash [Brca1L/L]) or conditional knock-in mouse lines (e.g., Gt(ROSA)26Sortm1(Pik3ca*H1047R)Egan<...

<ol>
	<li>Wagner, K. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cre-mediated+gene+deletion+in+the+mammary+gland.">Cre-mediated gene deletion in the mammary gland.</a> Nucleic Acids Research. 25 (21), 4323-4330 (1997).</li><li>Selbert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+BLG-Cre+mediated+gene+deletion+in+the+mammary+gland.">Efficient BLG-Cre mediated gene deletion in the mammary gland.</a> Transgenic Research. 7 (5), 387-396 (1998).</li><li>Jonkers, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+tumor+suppressor+activity+of+BRCA2+and+p53+in+a+conditional+mouse+model+for+breast+cancer.">Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.</a> Nature Genetics. 29 (4), 418-425 (2001).</li><li>van Bragt, M. P., Hu, X., Xie, Y., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RUNX1,+a+transcription+factor+mutated+in+breast+cancer,+controls+the+fate+of+ER-positive+mammary+luminal+cells.">RUNX1, a transcription factor mutated in breast cancer, controls the fate of ER-positive mammary luminal cells.</a> eLife. 3, e03881 (2014).</li><li>Tao, L., van Bragt, M. P., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Long-Lived+Luminal+Subpopulation+Enriched+with+Alveolar+Progenitors+Serves+as+Cellular+Origin+of+Heterogeneous+Mammary+Tumors.">A Long-Lived Luminal Subpopulation Enriched with Alveolar Progenitors Serves as Cellular Origin of Heterogeneous Mammary Tumors.</a> Stem Cell Reports. 5 (1), 60-74 (2015).</li><li>Xu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+mutation+of+Brca1+in+mammary+epithelial+cells+results+in+blunted+ductal+morphogenesis+and+tumour+formation.">Conditional mutation of Brca1 in mammary epithelial cells results in blunted ductal morphogenesis and tumour formation.</a> Nature Genetics. 22 (1), 37-43 (1999).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ETV6-NTRK3+fusion+oncogene+initiates+breast+cancer+from+committed+mammary+progenitors+via+activation+of+AP1+complex.">ETV6-NTRK3 fusion oncogene initiates breast cancer from committed mammary progenitors via activation of AP1 complex.</a> Cancer Cell. 12 (6), 542-558 (2007).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+loss+of+BRCA1+and+p53+in+mice+induces+mammary+tumors+with+features+of+human+BRCA1-mutated+basal-like+breast+cancer.">Somatic loss of BRCA1 and p53 in mice induces mammary tumors with features of human BRCA1-mutated basal-like breast cancer.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (29), 12111-12116 (2007).</li><li>Molyneux, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+basal-like+breast+cancers+originate+from+luminal+epithelial+progenitors+and+not+from+basal+stem+cells.">BRCA1 basal-like breast cancers originate from luminal epithelial progenitors and not from basal stem cells.</a> Cell Stem Cell. 7 (3), 403-417 (2010).</li><li>Koren, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PIK3CA+induces+multipotency+and+multi-lineage+mammary+tumours.">PIK3CA induces multipotency and multi-lineage mammary tumours.</a> Nature. 525 (7567), 114-118 (2015).</li><li>Van Keymeulen, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+multipotency+by+oncogenic+PIK3CA+induces+breast+tumour+heterogeneity.">Reactivation of multipotency by oncogenic PIK3CA induces breast tumour heterogeneity.</a> Nature. 525 (7567), 119-123 (2015).</li><li>Tao, L., Xiang, D., Xie, Y., Bronson, R. T., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+p53+loss+in+mouse+luminal+cells+causes+clonal+expansion+and+development+of+mammary+tumours.">Induced p53 loss in mouse luminal cells causes clonal expansion and development of mammary tumours.</a> Nature Communications. 8, 14431 (2017).</li><li>Tao, L., van Bragt, M. P. A., Laudadio, E., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+Tracing+of+Mammary+Epithelial+Cells+Using+Cell-Type-Specific+Cre-Expressing+Adenoviruses.">Lineage Tracing of Mammary Epithelial Cells Using Cell-Type-Specific Cre-Expressing Adenoviruses.</a> Stem Cell Reports. 2 (6), 770-779 (2014).</li><li>Sutherland, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+of+origin+of+small+cell+lung+cancer:+inactivation+of+Trp53+and+Rb1+in+distinct+cell+types+of+adult+mouse+lung.">Cell of origin of small cell lung cancer: inactivation of Trp53 and Rb1 in distinct cell types of adult mouse lung.</a> Cancer Cell. 19 (6), 754-764 (2011).</li><li>Shackleton, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+functional+mammary+gland+from+a+single+stem+cell.">Generation of a functional mammary gland from a single stem cell.</a> Nature. 439 (7072), 84-88 (2006).</li><li>The Cancer Genome Atlas Network. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+portraits+of+human+breast+tumours.">Comprehensive molecular portraits of human breast tumours.</a> Nature. 490 (7418), 61-70 (2012).</li><li>Nik-Zainal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+history+of+21+breast+cancers.">The life history of 21 breast cancers.</a> Cell. 149 (5), 994-1007 (2012).</li><li>Abba, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Molecular+Portrait+of+High-Grade+Ductal+Carcinoma+In+Situ.">A Molecular Portrait of High-Grade Ductal Carcinoma In Situ.</a> Cancer Research. 75 (18), 3980-3990 (2015).</li></ol>

The authors have nothing to disclose.

The success of this approach for inducing mammary tumors from different subpopulations of MECs relies not only on choosing appropriate cell-type-specific promoters (to drive Cre expression) but also on the intraductal injection procedure itself. The idea behind this approach is that the injected Ad-Cre viruses are retained in the ductal tree, which is a concealed structure with lumen, and therefore, only MECs are exposed to the viruses and are infected by Ad-Cre. Due to the limited lumen space within the mammary ducts, i...

The overall goal of this method is to develop a new breast cancer modeling approach based on an intraductal injection of Ad-Cre into the mouse MG. The Cre/loxP recombination-based genetic approach has been widely used to model human breast cancer in mice. The first generation of Cre/loxP-based breast cancer mouse models are generated by using Cre-expressing transgenic mice under the control of MEC-specific promoters (e.g., MMTV-Cre for luminal MECs and a portion of basal MECs, Wap-Cre and Blg-Cre for luminal progenitors and alveolar luminal MECs, K14-Cre for basal and a portion of luminal MECs1,2,3,4,5)6,7,8,9. However, while these Cre transgenic lines enable spatial control of Cre expression (i.e., in different subsets of MECs), they do not allow temporal control of Cre expression and Cre/loxP-mediated genetic manipulation. The second generation of Cre/loxP-based breast cancer mouse models utilize inducible Cre activity/expression approaches (e.g., use of Cre-estrogen receptor fusion [CreER], which can only induce Cre/loxP recombination upon administration of tamoxifen), and as a result, these genetic tools permit both spatial and temporal controls of the activation of oncogenic events in MECs (e.g., K8-CreER- and K5-CreER-based models)10,11,12. In both generations of breast cancer mouse models, as promoters used to drive Cre or CreER expression (e.g., Krt8, Krt5) may also be active in epithelial cells of other organs (i.e., they are cell-type-specific but not organ-specific) or have a leaky expression in cell types other than epithelial cells (e.g., MMTV, which has leaky activity in bone marrow hematopoietic cells), these approaches may lead to the development of unwanted cancer(s) in other organ(s). If these unexpected cancers cause lethality in the affected mice, the original purpose of modeling breast cancer in these mice may be prohibited (e.g., MMTV-Cre-driven oncogenic events may lead to hematopoietic malignancies and early death of the mice, due to leakiness of the MMTV promoter in hematopoietic cells)4.
Here we report a breast cancer modeling approach in mice that allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner. This approach is based on an intraductal injection of Ad-Cre into mouse MGs (and is, thus, organ-specific). Cre expression can be controlled by using different MEC subpopulation-specific promoters embedded in the adenoviral vector (e.g., Krt8 for luminal MECs, Krt5 for basal MECs, thus achieving cell-type specificity). Cancer induction in MGs can be temporally controlled by an injection of Ad-Cre into mice at different ages, starting from 3-4 weeks of age (pubertal) to the adult stage.

<table>
	<tbody>
		<tr>
			<td>33-gauge needle</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>point style 3 blunt</td>
		</tr>
		<tr>
			<td>7mm Reflex Clip</td>
			<td>Braintree Scientific</td>
			<td>RF7 CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenovirus, Ad-K5-Cre</td>
			<td>University of Iowa Viral Vector Core</td>
			<td>Ad5-bk5-Cre (VVC-Berns-1547)</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenovirus, Ad-K8-Cre</td>
			<td>University of Iowa Viral Vector Core</td>
			<td>Ad5mK8-nlsCre</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol</td>
			<td>Fisher</td>
			<td>HC800-1GAL</td>
			<td>Prepare to 70% in use</td>
		</tr>
		<tr>
			<td>biotinylated CD31</td>
			<td>eBiosciences</td>
			<td>13-0311-85</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated CD45</td>
			<td>eBiosciences</td>
			<td>13-0451-85</td>
			<td></td>
		</tr>
		<tr>
			<td>biotinylated TER119</td>
			<td>eBiosciences</td>
			<td>13-5921-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromophenol Blue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>CD24-AF-700</td>
			<td>BD Pharmingen</td>
			<td>564237</td>
			<td></td>
		</tr>
		<tr>
			<td>CD24-PE</td>
			<td>eBiosciences</td>
			<td>12-0242-83</td>
			<td></td>
		</tr>
		<tr>
			<td>CD29-APC</td>
			<td>eBiosciences</td>
			<td>17-0291-82</td>
			<td></td>
		</tr>
		<tr>
			<td>CD29-PE</td>
			<td>eBiosciences</td>
			<td>12-0291-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Remover Lotion</td>
			<td>Nair</td>
			<td></td>
			<td>9 Oz</td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton</td>
			<td>7636-01</td>
			<td>0.025 mL</td>
		</tr>
		<tr>
			<td>Iodophors</td>
			<td>Betadine</td>
			<td></td>
			<td>10% Povidone-iodine</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter</td>
			<td>NDC 10019-360-40</td>
			<td>1-2.5%</td>
		</tr>
		<tr>
			<td>Loxicam</td>
			<td>Norbrook</td>
			<td>NDC 55529-040-10</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Lubricant Eye Ointment</td>
			<td>Akorn</td>
			<td colspan="2">NDC 17478-062-35</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Pentair</td>
			<td>9M</td>
			<td>Watchmaker&#39;s Forceps</td>
		</tr>
		<tr>
			<td>Micro-dissecting tweezers</td>
			<td>Dumont</td>
			<td>M5</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq 5X Master Mix</td>
			<td>New England Biolabs</td>
			<td>M0285L</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling breast cancer via an intraductal injection of cre-expressing adenovirus into the mouse mammary gland

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.

Representative PCR genotyping results for the R26Y and Trp53L alleles are shown in Figure 1.
Although, in principle, all 10 MGs can be subjected to the intraductal injection procedure, practically, the two fourth inguinal MGs are typically selected for injection, due to their easier accessibility and larger MG sizes (Figure 2). During the su...

Watch this Scientific Journal Video about Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland at JoVE.com

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland

The goal of this protocol is to describe a new breast cancer modeling approach based on the intraductal injection of Cre-expressing adenovirus into mouse mammary glands. This approach allows both cell-type- and organ-specific manipulation of oncogenic events in a temporally controlled manner.

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are ...

This protocol will help researchers generate a new type of mouse model to recapitulate human breast cancer development from different subsets of mammary epithelial cells. This technique allows researchers to generate breast cancer mouse models in developmental stage tissue on a cell type specific manner and without a need for extensive mouse breeding. To begin this procedure, generate, maintain, and anesthetize floxed mice as described in the text protocol.Bring the mouse to an area separate from where the surgery is to be performed. After confirming anesthesia by performing a toe pinch, inject Meloxicam as analgesia subcutaneously at a dose of five milligrams per kilogram to prepare for the surgical procedure. Expose the nipple surgical site by applying several drops of hair removal cream.Remove excessive cream and loose hair using soft paper towels. Shaving is not recommended to avoid damage to the nipples. Disinfect the surgical site with iodophors first followed by 70%alcohol and end with a final application of scrub iodophors.Do this in a circular motion from the center of the work area toward the periphery using a gauze sponge or a cotton-tipped applicator. Repeat the cycle three to four times. Use aseptic techniques throughout the surgical procedure.Make an incision site on the skin at a length of approximately one centimeter between the two fourth inguinal mammary glands. Carefully separate the skin flap from the parietal peritoneum so as to visual the mammary ductal tree. Carefully hold the nipple with watchmakers'forceps to remove the exterior nipple without cutting any nearby skin using a microdissection scissor.Load approximately three to five microliters of Cre-adenovirus injection mixture into a 25 microliter Hamilton syringe with a 33 gauge metal hub needle affixed. Estimate the volume of the injection mixture in the syringe based on the blue dye included in the mixture. Gently hold the edge of the skin flap with a fine curved tweezer and inject the Cre-adenovirus injection mixture slowly into the nipple while monitoring the spreading of blue dye into the mammary ductal tree.Maintain the injection rate at slow as possible to avoid damage to the ductal lumen. A successful intraductal injection is indicated by injected fluids spreading throughout the entire ductal tree without leaking into the stroma compartment. Gently withdraw the needle from the nipple to avoid any leakage of the injected fluid.Examine the distal side of the mammary gland or the surrounding area of the injected nipple. Dye diffusing into the nearby stroma as shown here indicates a mammary fat pad injection rather than a successful intraductal injection. Close the surgical wounds in the skin with wound clips.Remove the mouse from the anesthesia and place it on a heating pad inside a clean cage for recovery. Monitor the development of the mammary tumor as described in the text protocol. The youngest female mice for which intraductal injection can be successfully performed are those at approximately three weeks of age.Although for most mammary tumor induction experiments, young adult female mice are used. Intraductal injection with a larger volume of the injection mixture can be performed in female mice during early to mid gestation to target alveolar cells. To target different mammary epithelial cell subpopulations for mammary tumor induction, Cre-adenoviruses under the control of different mammary epithelial cell subset specific promoters were used for injection.For example Cre-adenovirus under the control of keratin 8 promoter was used to target luminal mammary epithelial cells. Cre-adenovirus under the control of keratin 5 promoter targeted the basal lineage leading to genetic marking of only basal mammary epithelial cells. For a female P53 floxed homozygous mice with a ROSA26-YFP reporter, the intraductal injection of keratin 8 Cre-adenovirus led to the development of mammary tumors several months after the injection.Due to the inclusion of the conditional R26-Y reporter, tumor epithelial cells were typically marked by YFP and could be detected by flow cytometry. The cells can be enriched by the flow sorting of YFP positive cells for further analysis. The most important things to remember when attempting this procedure are to use a small injection volume, inject slowly, and also withdraw the needle slowly.Following this procedure, one can monitor progression of the targeted YFP positive mammary epithelial cells to pre-malignant and cancer stages by flow cytometry or co-immunofluorescence staining of the injected gland. This technique will pave the way for researchers to study cells of origin of breast cancer, how they respond to different autogenic events and how they progress to cancer cells. Both adenovirus and the needle for injection are potentially hazardous.Therefore, one should always wear gloves and use safety needles and syringes when performing intraductal injection.

modeling-breast-cancer-via-an-intraductal-injection-cre-expressing

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JoVE Journal

Cancer Research

12.4K vistas.  Brigham and Women's Hospital. Este protocolo ayudará a los investigadores a generar un nuevo tipo de modelo de ratón para recapitular el desarrollo del cáncer de mama humano a partir de diferentes subconjuntos de células epiteliales mamarias. Esta técnica permite a los investigadores generar modelos de ratón de cáncer de mama en el tejido del escenario del desarrollo de una manera específica de tipo celular y sin necesidad de una extensa cría de ratón. Para comenzar este procedimiento, genere, mantenga y anesthe...